Method of rapidly quantifying global DNA methylation

ABSTRACT

This invention is related to a method and assay kit for rapidly quantifying global DNA methylation through immobilizing DNA by simple dry-capture on the plastic carrier followed by immunodetection of 5-methylcytosine structure that is the marker of DNA methylation.

CROSS-REFERENCE TO RELATED APPLICATIONS

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STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

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REFERENCE TO A MICROFICHE APPENDIX

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BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention is related to a method and assay kit for rapidlyquantifying global DNA methylation through immobilizing DNA by simpledry-capture on the plastic carrier followed by immunodetection of5-methylcytosine structure that is the marker of DNA methylation.

2. Description of the Related Art

DNA methylation is an epigenetic modification which is catalyzed by DNAcytosine-5-methyltransferases (DNMTs) and occurs at the 5-position (C5)of the cytosine ring, within CpG dinucleotides. DNA methylation isessential in regulating gene expression in nearly all biologicalprocesses including development, growth, and differentiation (Laird P Wet al: Annu Rew. Genet, 1996; Reik W et al: Science, 2001; Robertson K Det al: Nature Rew. Genet, 2005). Alterations in DNA methylation havebeen demonstrated to cause the change in the gene expression. Forexample, hypermethylation leads to gene silencing or decreased geneexpression while hypomethylation activates the genes or increases geneexpression. Region-specific DNA methylation is mainly found in5′-CpG-3′dinucleotides within the promoters or in the first exon ofgenes, which is an important pathway for the repression of genetranscription in diseased cells. Global DNA hypomethylation is likelycaused by methyl-deficiency due to a variety of environmentalinfluences, and has been proposed as a molecular marker in multiplebiological processes such as cancer. It is well demonstrated that thedecrease in global DNA methylation is one of the most importantcharacteristics of cancer (Feinberg A P et al, Nature, 1983; Gama-Sosa MA et al, Nucleic Acids Res, 1983). Thus the determination of globalmethylation in cancer cells could provide very useful information forthe detection and analysis of this disease. Many methods for thedetection of global DNA methylation have been developed. These methodsinclude: (1) high-performance liquid chromatography or thin-layerchromatography analysis (Breter H J et al: J chromatogr, 1976; Wagner Iet al: Biochim Biophys Acta, 1981; Leonard S A et al: J chromatogr,1993). In the analysis, DNA is digested into single nucleotides andtotal genomic 5-methylcytosine is quantified; (2) immunohistochemicalstaining in which tissue section is stained with anti-5-methylcytosineantibody to detect 5-methylcytosine positive cells (Hemandez-Blazquez FJ et al: Gut, 2000; Piyaphilake C J et al: Dis Markers, 2005); (3) dotblot assay in which DNA is dot-blotted onto a nitrocellulose membranefollowed by immunodetection of methylcytosine (Oakeley E J et al: Proc.Natl. Acad. Sci. USA, 1997; Tao L et al: Toxic. Sci, 2005); (4) methylaccepting capacity or radiolabeled methyl incorporation (RMI) assay inwhich DNA is incubated with ³H-S-adenosylmethionine (³H-SAM) in thepresence of methylase and RMI is inversely related to the degree of DNAmethylation. (Wu J et al: Proc. Natl. Acad. Sci. USA, 1993; Belinsky S Aet. al: Proc. Natl. Acad. Sci. USA, 1996); (5) analysis of repetitiveDNA element methylation by PCR (Yang A S et al: Nucleic Acids Res,2004). However these methods are labor intensive, time-consuming, or arenot quantitative, or require large amounts of DNA (>250 ng) as thestarting material for measurement, or rely on the use of expensiveequipment and radioisotope reagents. These disadvantages lead tocost-ineffectiveness, low throughput and inconvenience for routineapplication in most health and medical institutions. Thus, there is aneed for establishing a method to improve the detection of global DNAmethylation.

BRIEF SUMMARY OF THE INVENTION

The present invention provides an improved method and kit which rapidlyquantify global DNA methylation through immunodetection of5-methylcytosine structure comprising the step of:

-   1) Isolation and purification of DNA from biological materials;-   2) Coating of the plastic carrier with the nucleic acid binding    substance;-   3) Immobilization of DNA by dry-capture on the plastic carrier which    is coated with the nucleic acid binding substance;-   4) Detection of 5-methylcytosine structure contained in the DNA with    the anti-5-methylcytosine specific antibody;-   5) Detection of anti-5-methylcytosine antibody with the secondary    antibody conjugated with label molecules;-   6) Fluorescent or color development of antibody conjugated with    label molecules and quantification of fluorescent or color    intensity.

Thus the invention allows a rapid quantification of global DNAmethylation to be achieved. The invention is based on the finding thatnucleic acid binding substances coated on the plastic surface can morerapidly and conveniently enhance DNA immobilization and retention on theplastic surface in a simple dry-capture manner at appropriatetemperature. The invention is also based on the finding that theimmunodetection of 5-methylcytosine can be quantitatively achievedthrough specific antibody recognition followed by color or fluorescencedevelopment and measurement. Therefore the method presented in thisinvention significantly overcomes the weaknesses existing in the priortechnologies and enables global DNA methylation to be quantified rapidlyand efficiently.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a diagram of the rapid process for quantifying global DNAmethylation. The process involves: (1) coating of plastic carrier withnucleic acid binding substance; (2) immobilization of DNA by dry-captureon the glass or plastic well which is coated with nucleic acid bindingsubstance; (3) detection of 5-methylcytosine structure contained in theDNA with the anti-5-methylcytosine specific antibody; (4) detection ofanti-5-methylcytosine antibody with the secondary antibody conjugatedwith label molecules; and (5) fluorescent or color development ofantibody conjugated label molecules and quantification of fluorescent orcolor intensity.

FIG. 2 shows the effect of different nuclei acid binding substances ondetection of global DNA methylation. The experiment was carried out asdescribed in Example 1.

FIG. 3 shows effect of different buffer on detection of global DNAmethylation. The experiment was carried out as described in Example 2.

FIG. 4 shows that the dry-capture more efficiently increase detection ofglobal DNA methylation. The experiment was carried out as described inExample 3.

FIG. 5 shows the quantification of global DNA methylation in normal andcancer cells. The experiment was carried out as described in Example 4.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method and kit for rapidly quantifyingglobal DNA methylation through immobilizing DNA by simple dry-capture onthe plastic carrier followed by immunodetection of 5-methylcytosinestructure that is the marker of DNA methylation. A basic outline of themethod presented in this invention is described in FIG. 1. This methodis particularly useful for rapidly completing a global DNA methylationassay in a short time. This method is also particularly useful forquantifying global DNA methylation in a high throughput format.

According to the method of this invention, DNA could be isolated bylysis of cells with lysis buffer containing a sodium salt, Tris-HCl,EDTA and detergents such as sodium dodecyl sulphate (SDS) orcetyltrimethylammonium bromide (CATB). Tissue fragments should behomogenized before lysing. For example, disaggregating of tissuefragments can be performed by stroking 10-50 times, depending on tissuetype, with a Dounce homogenizer. DNA can be further purified by mixingwith a high concentration of sodium chloride and then adding into acolumn pre-inserted with a silica gel, a silica membrane, or a silicafilter. The DNA that binds to the silica matrix is washed by adding awashing buffer and eluted with TE buffer or water. DNA can also beisolated and purified by using commercially available DNA extractionkits such as QiaAmp blood or tissue kits. The starting materials for DNAextraction can be from various species including, but not limited to,fresh tissues, frozen tissues, formalin fixed and paraffin embeddedtissues, body fluids, and cultured cells.

The purified DNA can be then immobilized on the plastic carrier which iscoated with nucleic acid binding substance. The carrier should bepolystyrene plastic with high hydrophobic properties and could be invarious sizes and forms including, but not limited to, 8-well strip,12-well strip, 16-well strip, 32-well strip, 48-well strip ormicroplate, 96-well microplate, 384-well microplate, 1516-wellmicroplate, microscopic slide, and microarray slide. A nucleic acidbinding substance can be selected from the epoxy silane, carbodiimide,dicyclohexylcarbodiimide, N-hydroxysuccinimide, p-nitrophenol andpoly-L-lysine. Preferably, poly-L-lysine is used for coating the plasticcarrier. Coating of the substance can yield a dense layer of aminegroups for initial ionic attachment of the negatively charged phosphategroups in the DNA backbone. The DNA can subsequently be attachedcovalently to the carrier. A 0.01% poly-L-lysine solution is prepared bydiluting 0.1% of poly-L-lysine solution (Sigma) with water and PBS atthe 1:10 ratios. For multi-well microplate or strips, 150 μl of 0.01%poly-L-lysine solution is added into the well and incubated at roomtemperature for 2 h. After washing with water, microplate or strips canbe dried at 37° C. or room temperature. For the plastic slide, 200 ml of0.01% poly-L-lysine solution is poured into glass slide box and theslide is then put in the box to soak at room temperature for 1 h. Excessliquid from the slide is removed by spinning the rack of the slide onmicrotiter plate carriers at 300-500 rpm. The slide is then dried at 40°C. for 20-30 min.

The plastic carrier coated with poly-L-lysine should be stored at least14 days to become highly hydrophobic and then can be used for DNAimmobilization. For multi-well microplate or strip, Purified DNA isdiluted with water or TE buffer at different concentrations and 10-50 μlof DNA solution is added into the well of the poly-L-lysine-coatedmulti-well microplate or strip. Preferably, 25-30 μl of DNA solution isadded into the well to just cover the bottom of the well. The multi-wellmicroplate or strip is incubated at 37° C. with no humidity for 2 hfollowed by incubating at 60° C. for 20 min to evaporate the solutionand dry the well. Preferably, the multi-well microplate or strip isincubated at 42° C. with no humidity for 1 h followed by incubating at60° C. for 30 min to accelerate dry of the well. For microscopic ormicroarray slide, 0.2-4 μl of DNA solution, depending on the requirednumber of spots, is added to each spot area. The slide can be incubatedat 37° C. with no humidity for 30 min followed by incubation at 60° C.for 5 min or incubation at 42° C. with no humidity for 30 min to dry thespotted area. DNA amount to be immobilized can be from 10 to 200 ng,preferably, from 50 to 100 ng, more preferably, 200 ng. A 200 ng of DNAamount would ensure the small fraction of methylated DNA containing inDNA to be detectable while still allowing high specificity to beachieved.

According to the method of the invention, the antibody specific to the5-methylcytosine is added and reacted with the 5-methylcytosinecontained in DNA immobilized on the plastic carrier. Before this step,DNA-immobilized plastic carrier should be blocked with blocking buffercontaining 1-3% BSA and phosphate salts. The blocking would prevent theunspecific binding of antibody and thus reduce the background of finaldetection. The blocking can be done by adding 2% BSA solution into thewell of multi-well microplate or strip, or by soaking the slide in the2% BSA solution at room temperature or 37° C. for 1-2 h. The plasticcarrier is then washed with the washing buffer comprising Tris-salineand 0.05% tween-20 preferably comprising phosphate saline and 0.1%tween-20. Once the washing is completed, the 5-methylcytosine antibodyis added to the plastic carrier. The antibody specific to the5-methylcytosine may include mouse monoclonal IgG, rat monoclonal IgG,rabbit polyclonal IgG, goat polyclonal IgG and sheep polyclonal IgG.According to the method of this invention, mouse monoclonal IgG ispreferable to use. The antibody can be unconjugated or conjugated withbiotin, or enzymatic label molecules such as horse radish peroxidase(HRP) and alkaline phosphotase (AP), or fluorescent label molecules suchas cy3, cy5, FITC, or gold label molecules, or quantum dot labelmolecules. The final concentration of the antibody added to the plasticcarrier should be 0.5-1 μg /ml. The plastic carrier is incubated at roomtemperature for 1 h or 37° C. for 45 min after adding antibody. Afterincubation, the plastic is washed with wash phosphate-saline containing0.1% tween-20 for 3 times. If the conjugated antibody is used, acolorimetric or fluorescent development can be directly carried outfollowed by signal measurement. If the unconjugated antibody is used, asecondary anti-mouse, or anti-rabbit, or anti-goat or anti-sheepantibody conjugated with label molecules is added to the plasticcarrier. The final concentration of secondary antibody can be from 0.01μg/ml to 0.5 μg/ml. The label molecules, depending on the requirement ofassay, include but are not limited to HRP, AP, biotin, fluorescein(FITC), Cy3, Cy5, rhodamine, dynabeads, texas red, Alexa fluor, BODIPY,captivate ferrofluid, cascade blue, beta-lactamase, marine blue,nanogold, Oregon green, pacific blue, and quantum dot. After washingwith phosphate-saline buffer containing 0.1% Tween-20, the methylatedDNA can be quantitatively detected through the colorimetric orfluorescent development. For colorimetric development, the solutioncontaining color-forming substrates is added to react with enzymaticlabel molecules such as HRP or AP to yield blue solution or deposit.Other suitable color-forming substrates will be apparent to personsskilled in the art. For fluorescent measurement, fluorescent intensityis directly detected with fluorescent spectrophotometer, fluorescentscanner, or fluorescent microscope.

In an assay, fully methylated DNA at 5-cytosine site by DNA methylasesuch as M.Sss I or NY-2A virus DNA which contains about 50% of5-methylcytosine can be used as the positive control. The water or TEsolution containing no DNA can be served as a negative control.

According to the invention, all of the components for DNA isolation,purification, coating substance of plastic carrier, substances for DNAimmobilization, and detection antibodies are commercially available Thisinvention also provides a kit containing all components required forrapid quantification of global DNA methylation in a multi-wellmicroplate/strip format. The kit includes: (a) an mouse monoclonalantibody specific for 5-methylcytosine structure and an anti-mouseantibody conjugated with HRP; (b) a microwell strip or microwell platecoated with poly-L-lysine for DNA immobilization; (c) the concentratedwashing buffer comprised of phosphate-saline and surfactants; (d) acolorimetric development solution containing color-forming substratespecific for HRP; (e) a positive control; and (h) an instruction forconducting an assay according to the method of this invention. In oneembodiment, the kit further comprises of selected components to meet therequirements for using different measurement equipments.

It has been discovered that the use of the method of this invention isable to drastically reduce the cost and time required for quantifyingglobal DNA methylaion. It has been also discovered that the use of themethod of this invention is able to allow global DNA quantification tobe much easer and more convenient than currently used methods, as themethod based on this invention can be carried out with regular equipmentsuch as a microplate reader or microscope. It has been furtherdiscovered that the use of the method of this invention enables thequantification of global DNA methylation to be performed in a highthroughput format and can be completed with excellent reproducibility.

The method of this invention for quantifying global DNA methylation isfurther illustrated in the following examples:

EXAMPLE 1

The experiment was carried out to compare the effect of differentnucleic acid binding substance coating on the detection of global DNAmethylation. Polystyrene 8-well strips were coated with 0.01%poly-L-lysine, or 1% polycarbodiimide, respectively. After storage at 4°C. for 2 weeks, the coated and uncoated strips were used for DNAimmobilization. 30 μl of naturally methylated NY-2A virus DNA atdifferent concentrations were added into the strip wells. The stripswere then incubated at 37° C. for 1.5 h followed by incubation at 60° C.for 30 min to dry the wells. The wells were blocked with 2% BSA solutionat 37° C. for 30 min followed by washing 3 times. 50 μl of monoclonalanti-5-methylcytosine antibody was then added at 0.5 pg/ml and incubatedat room temperature for 1 h. The wells were washed 3 times with PBScontaining 0.1% tween-20 after the antibody solution was removed. 50 μlof anti-mouse antibody conjugated with HRP at 0.1 μg/ml was added intothe wells and incubated at room temperature for 30 min. The wells werewashed 4 times with PBS containing 0.1% tween-20 after the antibodysolution was removed. 100 μl of the color development solutioncontaining TMB was added into the wells and wells were observed for 1-10min for blue color appearance. The 1 M HCl or H₂SO4 solution was addedto stop the color development and the optical density was measured witha microplate reader. As shown in the FIG. 2, the highest OD values ateach concentration point were observed for poly-L-lysine coated wells.

EXAMPLE 2

The experiment was carried out to compare the effect of dry-capturemethod and conventional immobilization methods on the detection ofglobal DNA methylation. Polystyrene 8-well strips were coated with 0.01%poly-L-lysine. After storage at 4° C. for 2 weeks, the coated stripswere used for DNA immobilization. With the method of this invention,naturally methylated NY-2A virus DNA was diluted with 1× TE buffer. 30μl of diluted virus DNA at different concentrations were added into thestrip wells. The strips were then incubated at 37° C. for 1.5 h followedby incubation at 60° C. for 30 min to dry the wells. With theconventional method, 50 μl of diluted virus DNA at differentconcentrations were added into the wells and incubated at roomtemperature for 4 h or at 4° C. overnight. The remaining solution wasremoved after incubation. The DNA-immobilized wells with both methodswere blocked with 2% BSA solution at 37° C. for 30 min followed bywashing 3 times. 50 μl of monoclonal anti-5-methylcytosine antibody wasthen added at 0.5 μg/ml and incubated at room temperature for 1 h. Thewells were washed 3 times with PBS containing 0.1% tween-20 after theantibody solution was removed. 50 μl of anti-mouse antibody conjugatedwith HRP at 0.1 μg/ml was added into the wells and incubated at roomtemperature for 30 min. The wells were washed 4 times with PBScontaining 0.1% tween-20 after the antibody solution was removed. 100 μlof the color development solution containing TMB was added into thewells and wells were observed for 2-10 min for blue color appearance.The 1 M HCl or H₂SO4 solution was added to stop the color developmentand the optical density was measured with a microplate reader. As shownin FIG. 3, the higher OD values at each concentration point wereobserved for the wells in which DNA was immobilized by dry-capturemethod than those by conventional methods.

EXAMPLE 3

The experiment was carried out to examine the detection sensitivity ofthe method based on this invention on the quantification of global DNAmethylation. Polystyrene 8-well strips were coated with 0.01%poly-L-lysine. After storage at 4° C. for 2 weeks, the coated stripswere used for DNA immobilization. Naturally methylated NY-2A virus DNAwas diluted with 1× TE buffer to different concentrations. 30 μl ofdiluted virus DNA were added into the wells at the amounts of 2 ng-100ng/well. The strips were then incubated at 37° C. for 1.5 h followed byincubation at 60° C. for 30 min to dry the wells. The wells were thenblocked with 2% BSA solution at 37° C. for 30 min followed by washing 3times. 50 μl of monoclonal anti-5-methylcytosine antibody was then addedat 0.5 μg/ml and incubated at room temperature for 1 h. The wells werewashed 3 times with PBS containing 0.1% tween-20 after the antibodysolution was removed. 50 μl of anti-mouse antibody conjugated with HRPat 0.1 μg/ml was added into the wells and incubated at room temperaturefor 30 min. The wells were washed 4 times with PBS containing 0.1%tween-20 after the antibody solution was removed. 100 μl of the colordevelopment solution containing TMB was added into the wells and wellswere observed for 2-10 min for blue color appearance. The 1 M HCl orH₂SO4 solution was added to stop the color development and the opticaldensity was measured with a microplate reader. The OD values could beobserved for the wells which contain as low as 1 ng of methylated DNA.

EXAMPLE 4

The experiment was carried out to examine the effect of the method basedon this invention on the quantification of global DNA methylation innormal and cancer cells. Polystyrene 8-well strips were coated with0.01% poly-L-lysine. After storage at 4° C. for 2 weeks, the coatedstrips were used for DNA immobilization. Genomic DNA isolated from MCF-7and HCT-116 cancer cells and from peripheral blood lymphocytes (PBL) wasdiluted to 8 μg/ml and 25 μl (200 ng) of each genomic DNA were addedinto the strip wells. The strips were then incubated at 37° C. for 1.5 hfollowed by incubation at 60° C. for 30 min to dry the wells. An equalamount (200 ng) of naturally methylated virus DNA was used as thepositive control. The wells were then blocked with 2% BSA solution at37° C. for 30 min followed by washing 3 times. 50 μl of monoclonalanti-5-methylcytosine antibody was then added at 0.5 μg/ml and incubatedat room temperature for 1 h. The wells were washed 3 times with PBScontaining 0.1% tween-20 after the antibody solution was removed. 50 μlof anti-mouse antibody conjugated with HRP at 0.1 μg/ml was added intothe wells and incubated at room temperature for 30 min. The wells werewashed 4 times with PBS containing 0.1% tween-20 after the antibodysolution was removed. 100 μl of the color development solutioncontaining TMB was added into the wells and wells were observed for 2-10min for blue color appearance. The 1 M HCl or H₂SO4 solution was addedto stop the color development and the optical density was measured witha microplate reader. The higher O.D values for PBL DNA were observed.

1. A method of rapidly quantifying global DNA methylation comprising: a)immobilization of DNA with a binding buffer onto a plastic carriercoated with poly-L-lysine at concentration of 0.001-0.1% by two-phasetemperature for said an appropriate period. b) immunodetection of5-methylcytosine structure contained in the DNA. 2-8. (canceled)
 9. Themethod according to claim 1 wherein said buffer is tris-EDTA solution.10. The method according to claim 1 wherein said two-phase temperatureis consisting of first phase at 25-42° C. followed by second phase at50-75° C.
 11. The method according to claim 1 wherein said two-phasetemperature is consisting of first phase at 37° C. followed by secondphase at 60° C.
 12. The method according to claim 1 wherein said anappropriate period is from 2 to 4 hours. 13-19. (canceled)
 20. Themethod according to claim 1 wherein said the kit is comprised of: a) themicrowell plate or strips coated with poly-L-lysine. b) the concentratedwashing buffer comprising of phosphate salt and 0.1% tween-20. c) theDNA immobilization buffer comprising of Tris-HCL and EDTA. d) theblocking buffer comprising of phosphate salt and 2% BSA e) the colordevelopment solution comprising of TMB and H₂O₂ f) the stop solutioncomprising of HCl g) a positive control from the enzymaticallymethylated genomic DNA or naturally methylated virus DNA h) instructionsfor conducting an assay according to the method of this invention